Prenatal screening for Down&#39;s syndrome using hyperglycosylated gonadotropin

ABSTRACT

A prenatal screening method for Down&#39;s syndrome involves assaying for hyperglycosylated gonadotropin in biological test samples such as urine, plasma or serum obtained from pregnant women. Hyperglycosylated gonadotropin comprises a variant population of chorionic gonadotropin, chorionic gonadotropin-free β-subunit, β-core fragment, and/or free α-subunit exhibiting differences in the carbohydrate content from what is observed in samples obtained from pregnant women carrying normal fetuses. Qualitative or quantitative observation of differences in the carbohydrate content of the hyperglycosylated gonadotropin population from corresponding control samples containing a normal gonadotropin population, or direct observation of the variant species seen in Down&#39;s syndrome, indicates that the woman&#39;s fetus has Down&#39;s syndrome. Typical screens involve carbohydrate analyses, immunoassays, or combinations of these methods. Some embodiments employ a lectin such as concanavalin A reactive to the carbohydrate moiety; others employ antibodies to at least one hyperglycosylated gonadotropin species.

RELATED APPLICATION DATA

This application claims priority benefit of U.S. patent application Ser.No. 60/025,568, filed Sep. 6, 1996, and PCT application numberPCT/US97/16657, filed internationally Sep. 5, 1997.

TECHNICAL FIELD

This invention relates to a Down's syndrome screening test for pregnantwomen.

A triple screen of α-fetoprotein, chorionic gonadotropin andunconjugated estrogen in serum has been suggested for the prenataldiagnosis of Down's syndrome (Bennett, J. C., and Plum, F., Cecil'sTextbook of Medicine, W. B. Saunders, Philadelphia, 1996, p. 165).However, it allows detection of only 60 to 65% of fetuses with thegenetic disorder and gives 5% false positive results. It is also limitedto the second trimester of pregnancy (15 to 24 weeks of gestation), andhas become expensive as significant license fees are being levied onlaboratories running human chorionic gonadotropin analyses usingconventional methods (Auxter, S., Clin. Labor News 23: 1-3 (1997)).

Definitive prenatal diagnosis of fetal chromosome abnormalities leadingto Down's syndrome, which affect 1 in 700 live births, typicallyinvolves instead culture of amniocytes at midtrimester gestation. Theprocedure involves the aspiration of a small sample of amniotic fluid(amniocentesis), culturing of the fetal cells contained in the fluid,and determination of the karyotype of these cells and thus the fetus.The major indications for the use of this technique for the detection ofchromosome abnormalities are maternal age (usually offered to allmothers over the age of 35 at the time of expected delivery), thepresence of a parental chromosome abnormality, or a maternal history ofcarrying a previous trisomic child or aborted fetus karyotyped to betrisomic. Direct transcervical aspiration of chorionic villi (chorionicvillus sampling, or CVS) has also been used for prenatal diagnosis.

Though both procedures have been shown to be relatively safe andreliable, it is generally accepted that they involve some risk,including risk of miscarriage, and, in the case of CVS, also risk oflimb hypoplasia in babies born following the procedure. It would bedesirable to have other methods for screening pregnant women for Down'ssyndrome fetuses, particularly screens that are noninvasive andsensitive. Most Down's syndrome cases occur in younger pregnant women,those under 35 at the time of expected delivery, or the majority ofpregnancies. Less invasive screening tests are needed employing serum orurine samples to identify those at high risk for Down's syndromepregnancies, who may not want the risk of amniocentesis or CVS.

BACKGROUND OF THE INVENTION

Human chorionic gonadotropin (hCG) is a glycoprotein hormone secreted inrelatively large quantities by the trophoblast cells of the placenta(Masure, H. R., et al., J. Clin. Endocrinol. Metab. 53: 1014-1020(1981)). hCG is composed of two dissimilar subunits, α (92 amino acidsand two N-linked oligosaccharides) and β (145 amino acids and twoN-linked and four O-linked oligosaccharides), joined noncovalently, andis detected in the serum and urine of pregnant women and in those withtrophoblast disease (hydatidiform mole and choriocarcimoma). Free α- andfree β-subunits, and degraded hCG and free β-subunit molecules are alsodetected in serum and urine samples (Birken, S., et al., Endocrinology122: 572-583 (1988)). The degraded molecules include nicked hCG andnicked free β-subunit, each cleaved between β-subunit residues 47 and 48(or less commonly between residues 43 and 44 or 44 and 45), nickedβ-subunit missing all or part of the C-terminal sequence (β93-145) and,a terminal degradation product comprising two fragments, β-subunitsequences 6-40 and 55-92, held together by disulfide linkages, foundprimarily in urine samples (FIG. 1). The terminal degradation producthas no O-linked oligosaccharides and degraded N-linked oligosaccharidemoieties, one or two N-linked pentasaccharides, versus twoundecasaccharides on free β-subunit and hCG (FIG. 2A). The terminaldegradation product was called β-core fragment (β-core, Blithe, D., etal., Endocrinology 122: 173-180 (1988)), firstly because of its smallsize (˜9,000 daltons; hCG is 37,000 daltons), and secondly because ofits retention of hCGB radioimmunoassay or β-submit core antisera (versusC-terminal or other) immunoreactivity (Birken, et al., and Masure, etal., cited above). Through most of a pregnancy, β-core fragment is theprincipal hCG β-subunit-related molecule in urine samples.

Serum and urine free β-subunit derive from three sources: direct sectionby trophoblast cells, slow dissociation of circulating hCG into free α-and β-subunits, and by the nicking of hCG by macrophage or neutrophilenzymes associated with trophoblast tissue, and the more rapiddissociation of nicked hCG in circulation (FIG. 1). Free β-subunit maybe nicked by nicking enzymes in the circulation. Urine β-core fragmentappears to derive from the degradation of nicked free β-subunit in thekidney.

In the late 1980s, the triple marker test mentioned above was developedto screen for Down syndrome pregnancies. It combined maternal age withserum measurements of hCG, α-fetoprotein, and unconjugated estriol(Bogart, M. H., et al., Prenat. Diagn. 7:623-630 (1987), U.S. Pat. No.4,874,693 to Bogart, Wald, N. J., et al., Br. J. Obstet. Gynaecol. 95:334-341 (1988), and Canick, J. A., J. Clin. Immunoassay 13: 30-33(1990)). More recently, serum-free β-subunit tests and freeβ-subunit-α-fetoprotein combinations have been introduced as alternativeDown syndrome-screening methods (Macri, J. N., el al., Am. J. Obstet.Gynecol. 163: 1248-1253 (1990) and Spencer, K., et al., Ann. Clin.Biochem. 30: 394-401 (1993)). The best serum free β-subunit combination,or the optimal triple marker test, however, detects only 60 to 65percent of Down's syndrome cases, with a 5 percent false-positive rate.At these detection and false-positive rates, approximately 80amniocenteses need to be performed to detect a single case of Downsyndrome, and a significant number of Down's syndrome cases are missed(Cole, L. A., et al., Prenatal Diagnosis 17: 607-614 (1997)). There is aneed for improvement in prenatal screening methods.

SUMMARY OF THE INVENTION

It is an object of the invention to provide a prenatal screening testfor Down's syndrome pregnancies.

It is another and more specific object of the invention to provide asensitive, noninvasive test for Down's syndrome fetuses in pregnantwomen.

It is a further object of the invention to provide an improvement in thetriple marker test that exhibits a decreased false positive rate.

These and other objects are accomplished by the present invention, whichprovides a novel diagnostic method for screening for the presence orabsence of Down's syndrome in the fetus of a pregnant woman whichcomprises obtaining a biological test sample from the woman anddetermining the presence of Down's syndrome by observation ofhyperglycosylated gonadotropin in the sample. This typically involvesmeasuring the concentration of hyperglycosylated gonadotropin, its freeβ-subunit, its free α-subunit, and/or its β-core fragment in the testsample, and determining the presence of Down's syndrome by observationthat the concentration in the test sample population differs from anormal hyperglycosylated gonadotropin or free α-subunit, free β-subunit,or β-core fragment population and/or is the same as, or similar to, aDown's syndrome population. In preferred embodiments, the test sample isurine, saliva, plasma or serum, the population compriseshyperglycosylated hCG, β-core fragment, free α-subunit, free β-subunit,and mixtures thereof, and any differences between the propertiesobserved in the normal and Down's syndrome samples reflect differencesobserved in the carbohydrate content of the glycopolypeptides and/orglycopeptides.

Carbohydrate compositional or structural analyses, immunoassays, andcombinations of these methods are generally employed. In someembodiments, hyperglycosylated gonadotropin is determined directly byassay for at least one hyperglycosylated species, i.e., variant hCG,free β-subunit, free α-subunit, and/or β-core fragment. These screenstypically employ a monoclonal, polyclonal, or fusion phage antibody to ahyperglycosylated or carbohydrate-variant hCG species in an ELISA,Western blot, or the like. In another embodiment, elevated levels ofmonosaccharides are observed in samples positive for Down's syndrome.Other screens employ lectins that bind the carbohydrate moieties,chromatography, chemical or electrophoresis or isolelectric focussingtests that detect glycosylation variants of hCG. Lectins may also beemployed to separate and/or concentrate hCG species having aberrantcarbohydrate moieties prior to immunoassay.

DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic line drawing illustrating the structures of hCGrelated molecules. Thick lines represent the peptide backbone, numbersindicate amino acid positions, and thin lines indicate sites ofdisulfide linkages. The letters indicate monosaccharides inoligosaccharide side chains. S=sialic acid; G=galactose;A=N-acetylglucosamine; M=mannose; and L=N-acetylgalactosamine.

FIG. 2 shows schematic drawings of N-linked oligosaccharides. Thestructures in FIG. 2A illustrate the N-linked oligosaccharides on normalpregnancy hCG and its free β-subunit, and free α-subunit. The FIG. 2Bstructure shows a degraded N-linked oligosaccharide on a normalpregnancy β-core fragment. The FIG. 2C structure indicates the O-linkedoligosaccharides on normal pregnancy hCG as its free β-subunit.Abbreviations used are Man for mannose, Gal for galactose, GlcNAc forN-acetylglucosamine, Fuc for fucose, and GalNAc forN-acetylgalactosamine. Variables are indicated by the ±.

FIG. 3 illustrates hyperglycosylated N-linked and O-linkedoligosaccharides on abnormal hCG species found elevated in Down'ssyndrome and/or related pathological conditions such as choriocarcinoma.FIG. 3A shows N-linked oligosaccharides on hyperglycosylatedgonadotropin and its free β-subunit and free α-subunit. FIG. 3B showsN-linked oligosacchrides on hyperglycosylated gonadotropin β-corefragment. FIG. 3C shows O-linked oligosacchrides on hyperglycosylatedgonadotropin and its free β-subunit. Abbreviations are the same as thatemployed in FIG. 2.

FIG. 4 is a plot of monoclonal B152 reactivity in an immunoassay forhyperglycosylated hCG in urine samples taken at the periodic intervalsindicated from 130 women pregnant with normal fetuses () and 9 womenpregnant with Down syndrome fetuses (◯). Medians were determined, andsamples, expressed as multiples of the normal pregnancy medians. A logGaussian line was fitted for the normal pregnancy medians adjusted forgestational age (50th centile). The 95th centile was also determined.

DETAILED DESCRIPTION OF THE INVENTION

This invention is based upon the observation of aberrant carbohydrateprofiles in hCG species (hyperglycosylated gonadotropin) obtained fromthe serum or urine of pregnant women carrying Down's syndrome fetuses,but not from women carrying normal fetuses.

In the practice of the invention, the presence or absence of Down'ssyndrome in the fetus of a pregnant woman is determined by a methodcomprising obtaining a biological test sample from the woman andassaying for hyperglycosylated gonadotropin in the sample. Thistypically involves a determination of whether the composition orphysical properties of the chorionic gonadotropin population in the testsample differs from a corresponding control sample containing a normalchorionic gonadotropin population and/or that the sample containselevated levels of at least one species of the hyperglycosylatedgonadotropin population observed in Down's syndrome.

As used herein the term “chorionic gonadotropin population” includeschorionic gonadotropin, α-subunits, β-subunits, β-core fragments, andmixtures thereof, and specifically includes variants of these speciesthat have abnormal monosaccharide compositions in their oligosaccharidemoieties or are hyperglycosylated such as those observed in Down'ssyndrome hCG populations. The term “hyperglycosylated gonadotropin”generically encompasses these latter species within the chorionicgonadotropin population, comprising hyerglycosylated gonadotropin,nicked gonadotropin, α-subunits, β-subunits, β-core fragments, andmixtures of any of these which exhibit aberrant carbohydrate profilesand/or aberrant carbohydrate levels as compared to normal levels.

hCG and β-core fragments are employed in many embodiments. It is anadvantage of the invention that pregnancy serum contains largequantities of hCG. hCG accounts for over 99% of the totalβ-immunoreactivity in pregnancy serum. Likewise, pregnancy urine has alarge population of β-core fragment. Indeed, the β-core population canaccount for as much as 70% of the total β-immunoreactivity in pregnancyurine (Blithe, et al., cited above). Thus, in preferred embodiments, theinvention provides a screening method for the abundant species, hCG inserum and β-core fragment in urine, and ones that are readily detectedusing standard techniques including immunoassays and the like clinicalmeasurements described by Birkin, et al., and Blithe, et al., citedabove and illustrated hereafter, or, because of variations observed inthe carbohydrate portion of Down's syndrome hCG species, carbohydrateanalyses such as use of a lectin specific to the carbohydrate, or use ofmonosaccharide compositions tests, electrophoresis or isoelectricfocussing to detect glycosylation variants. Some variant speciesobserved in Down syndrome and related aberrant pregnancies areillustrated in FIG. 3 and described in Elliott, M. M., et al., EndocrineJ., vol. 7 (1997). Note that many hyperglycosylated Down syndrome hCGspecies have, instead of a sialyllactosamine (NeuAc-Gal-GlcNAc)biantennary structure attached to a mannose core in the hCG β-subunit(FIG. 2), triantennary structures having an additional sialyllactosamine(FIG. 3A). Other hyperglycosylation features include increased levels offucose-containing (versus fucose-free) N-linked oligosaccharides, andhexasaccharide structures at the β-subunit O-linked carbohydrate sites(FIG. 3C). Hyperglycosylation is thus significantly elevated in Downsyndrome hCG species.

Any biological sample can be employed in methods of the invention,including, but not limited to, urine, saliva, serum, plasma, tears, andamnionic fluid. Saliva, urine, plasma or serum are preferred becausesamples are more voluminous and sampling involves no risk of harm to thefetus. Urine obtained from a pregnant woman in her first (generallydefined as about 9 weeks to 13 weeks, 6 days) or second trimester(generally defined as about 14 to 28 weeks) and is particularlypreferred in some embodiments. It is an advantage of the invention thatsamples can be analyzed in the first trimester, earlier in the pregnancythan previously described methods for assessing placental dysfunctionsuch as that disclosed in U.S. Pat. No. 4,874,693 cited above (18 to 25weeks). A screening method suitable for use from the 11th to the 19thweeks of gestation, for example, is illustrated in the Examples thatfollow and in FIG. 4.

Down's syndrome screens of the invention generally employ carbohydrateanalyses, immunoassays, or combination of these methods for detection ofhyperglycosylated gonadotropin, but any assay that functions toqualitatively or quantitatively determine variations in sampleconcentrations of hyperglycosylated gonadotropin from normal levels,and/or detects abnormal carbohydrate hCG moieties in the sample'sgonadotropin population can be employed in the practice of theinvention. Direct assay for at least one member of the variant Down'ssyndrome chorionic gonadotropin population is preferred. Some screensemploy lectins that assay for the carbohydrate moieties, chromatography,chemical or electrophoresis or isoelectric focussing tests that detectglycosylation variants of hCG, and/or antibodies to hyperglycosylated orcarbohydrate-variant hCG.

Immunoassays include, but are not limited, to assays employing specificantibodies to hyperglycosylated gonadotropin generated by standardmeans, and assays employing nonspecificly defined antibodies obtained byblind injections of Down's syndrome hCG or choriocarcinoma hCG into testanimals using standard methods. Any type of fusion phage, monoclonal orpolyclonal antibodies can be used in immunoassays of the invention, solong as the antibodies can be used in a reproducible fashion as markersfor variant Down's syndrome hyperglycosylated hCG species withoutrecognizing normal species, or as measures of the different levelsobserved in normal and variant populations, and specifically includeantibodies to the variant carbohydrate portion of the fragments. Anantibody that recognizes nicked hyperglycosylated hCG obtained from achoriocarcinoma patient but does not detect normal hCG, denominated asB152, is employed in an immunoassay described hereafter.

In a typical immunometric assay of the invention, an antibody specificfor a hyperglycosylated variant such as B152 is employed as captureantibody with a second labelled antibody to hCG, β-core fragment,α-subunit, and/or β-subunit to provide the assay with its polypeptidespecificity. The label on the second antibody comprise any chemical,radioactive, lanthanide, colored dye, or genetic tag used inenzyme-linked immunosorbent assays (ELISAs), Western blots, and othersensitive and specific immunoassays and immunoradiometric assays usingknown methodology. These include conjugating the antibody withhorseradish peroxidase or alkaline phosphatase that are easilymeasurable, typically using colorimetric, fluorometric or luminescentsubstrates. Genetic labels include firefly luciferase, employed becauseluciferase produces a bioluminescent molecule when incubated with itssubstrate, luciferin. Alternate embodiments employ a third antibody todetect the presence of the other antibodies.

Other embodiments employ the peptide-specific antibody as a captureantibody, and antibody specific to hyperglycosylated orcarbohydrate-variant gonadotropin and/or an abnormal carbohydrateportion thereof comprises the second labelled antibody in any of theimmunoassays described above. Competitive immunoassays employingantibodies such as B152 may also be employed to competitively detecthyperglycosylated gonadotropin. Alternate embodiments using concanavalinA or other carbohydrate-specific lectin can be used in place of thecapture antibody or labelled antibody. Some embodiments employ a lectinor chromatographic method to extract carbohydrate-variant hCG prior toan immunoassay.

Carbohydrate analyses include qualitative observations of differences inphysical properties between normal and Down's syndrome hCG populationsdescribed in the Examples hereafter, carbohydrate identification usingplant lectins specific to the variant carbohydrate portion of Down'ssyndrome hCG obtained by standard lectin screening methods, or any otherfingerprinting technique including qualitative or quantitativecarbohydrate composition analyses. An example employing concanavalin Aattached to a solid support, which binds the three mannose unit in thecarbohydrate portion of hCG (FIG. 2), is employed in an embodimentillustrated hereafter, but any lectin that exhibits differential bindingof hyperglycosylated versus normal hCG species can be employed inmethods encompassed by the invention.

In one embodiment, for example, the presence or absence of Down'ssyndrome in the fetus of a pregnant woman is determined in a methodwhich comprises obtaining a urine, saliva, plasma or serum test, sample,preferably serum or urine, from the woman, determining the carbohydratecontent of the hCG in the test sample, comparing the carbohydratecontent so obtained to the carbohydrate content of a correspondingcontrol sample containing a normal hCG population, and determining thepresence of Down's syndrome by observation that the carbohydrate contentof the hCG population differs from the control sample. An observedincrease of at least about 25%, preferably at least about 50%, in thelevel of at least one hyperglycosylated species is preferred. Theobservation of aberrant species not observed at all in normal hCGsamples such as those set out in FIG. 3 is particularly preferred.

In one embodiment of the method, the presence of Down's syndrome isdetermined by observation that the monosaccharide content in the testsample is elevated in comparison to the control sample. This can involvean observation of differences in elution patterns such as that describedbelow, or an analysis for N-acetylglucosamine. An increase of at leastabout 50% is observed in typical analyses.

Alternatively, the properties of the chorionic gonadotropin populationin a sample are determined using electrophoresis, isoelectric focussingtests that detect glycosylation variants of hCG, chromatography, andmixtures of these techniques. Chromatographic methods encompass thoseusing hydrophobic interactions or other ligand chromatography, such asthat employing Blue Dextran Sepharose® (an agarose hydrophobicchromaography gel that adsorbs different proteins according to theiraffinity for blue dextran) illustrated hereafter, but any method ofcomparing physical properties of the glycopolypeptides or glycopeptidescan be employed, e.g., simple qualitative observation of differences inelution patterns. These include, but are not limited to, columnchromatography, coated beads, coated test tubes or plates, differentialbinding, extractions, and the like, and combinations of thesetechniques. It is an advantage of the invention that where β-corefragments are assayed, the markers are small and soluble.

Normal chorionic gonadotropin populations used as controls in manyscreening methods of the invention can be obtained or cloned from womencontaining normal karotype fetuses, or obtained commercially. hCG β-corefragments, for example, can be extracted from hCG preparations obtainedfrom Organon, Diosynth Division (Oss, Holland) as described by Birken,et al., cited above.

Screening methods of the invention can be used alone, or in combinationwith other screening methods. Other screening methods include, but arenot limited to, unconjugated and/or conjugated estriol measurements, hCGassays, β-core fragment analyses, free β-subunit or free α-subunitanalyses, PAPP-A or CA125 analyses, α-fetoprotein analyses, inhibinassays, observations of fetal cells in serum, and ultrasound. It is anadvantage of the invention that the method can replace the hCGdeterminations currently employed in the triple marker test describedabove, thereby improving its sensitivity and reliability. As mentionedpreviously, employing a method of the invention as a substitute for theconventional hCG assays in the triple marker or other test provides theadded advantage of an early assay for Down syndrome because thehyperglycosylated hCG screens can be used in the first trimester ofpregnancy. With an early diagnosis, the woman has the option ofterminating her pregnancy early by non-surgical methods, with minimalmortality or fertility loss.

EXAMPLES

The following are presented to further illustrate and explain thepresent invention and should not be taken as limiting in any regard.

Example 1

Structural handles were sought to differentiate between Down syndromeand normal pregnancy β-core fragment. Samples of β-core fragment werepurified from the urine of 6 women, 3 having normal karyotype and 3 Downsyndrome second trimester pregnancies, as well as two samples from 2diabetic patients with normal karyotype. β-Core fragments were extractedwith 2 volumes of acetone at 4° C. The acetone precipitates werecollected, dried, and taken up in phosphate-buffered saline (PBS). Thesamples were applied to a column of Blue Dextran-Sepharose™ (Pharmacia)washed with PBS, and then eluted consecutively with PBS containing 0.6 MNaCl and then PBS containing 1.0 M NaCl. β-Core fragment levels wasmeasured in the eluates. The 3 Down's syndrome β-core fragment samplesall eluted from the Blue Dextran column with the PBS containing 0.6 MNaCl, while the 5 normal karyotype samples eluted from the same columnsin the next step, with PBS containing 1.0 M NaCl. This shows adifference in the physical properties of Down syndrome β-core fragment.

N-terminal peptide sequence analysis was conducted on the purifiedfragments obtained from 2 of the individual Down's syndrome β-corefragment samples, 1 individual control β-core fragment sample, and 1β-core fragment sample purified from a pool of control urine. Thepurified samples all yielded an N-terminal peptide sequence identical tothat described by Birken, et al., cited above. The individual and pooledcontrol samples had a carbohydrate composition (3 mannose, 0.5 fucose,and 2 N-acetylglucosamine residues) identical to those described byBlithe, et al,. cited above, for other normal karyotype β-core fragmentsamples. (See structure FIG. 2B. N-Acetyl glucosamine content wasdetermined as the hydrolysis product, glucosamine.) The 2 Down'ssyndrome samples, however, exhibited somewhat different compositions,with notably more N-acetylglucosamine residues (3 mannose, 1 fucose and3.5 N-acetylglucosamine residues, and 3 mannose, 1 fucose, and 3.2N-acetylglucosamine residues, respectively; see structure FIG. 3B). Asshown in FIG. 1, β-core fragments are derived from the β-subunit of hCG.If the carbohydrate moieties on the variant Down's syndrome β-corefragment contain additional N-acetylglucosamine residues, it would beexpected that β-subunit of hCG from which β-core fragment is derivedwould also contain different amounts of N-acetylglucosamine and likelyincreased amounts of sialyllactosamine antennae, depending on the modeof degradation of the subunit. The aberrant carbohydrate composition ofDown's syndrome β-core fragment explains the aberrant elution profilefrom Blue Dextran Sepharose™.

Example 2

The binding of urine β-subunit to agarose-bound concanavalin A (Con A)was examined. Con A binds oligosaccharides and glycoproteins withbiantennary-type N-linked oligosaccharides like found on normalpregnancy hCG. Glycoproteins may be released from Con A with acompetitive inhibitor, α-methylmannoside. Molecules with triantennaryoligosaccharides, e.g., hyperglycosylated hCG, poorly bind Con A. Hence,Con A binding was employed to screen for Down's syndrome pregnancies.

Con A-agarose, 0.15 ml, was placed in 1.5 ml conical centrifuge tubes,0.5 ml of urine was added plus 0.5 ml PBS, pH 7.3 buffer. Tubes wererocked for 15 minutes, and then spun at 500×g to settle the gel. Theunbound urine-PBS was then removed. Gels were then washed with 1 ml PBS.The wash released loosely bound proteins. The tubes were again rockedand spun, and the wash was removed. Con A was then specifically elutedwith 1 ml PBS containing 0.05 M α-methylmannoside. Again tubes wererocked and spun, and the eluate was removed. The wash and eluate werethen tested for free β-subunit using an immunoassay.

While the bulk of urine free β-subunit was bound and only elutable withα-methylmannoside small but varying component was loosely bound andextracted in the PBS wash. Free β-subunit (>0.5 ng/ml) was detected in18 of 109 (17%) control samples and 9 of 15 (60%) Down syndrome samplewashes. Higher levels of free β-subunit (>1.7 ng/ml) were found in 9 outof 109 (8%) control sample and 7 of 15 (47%) Down syndrome samplewashes. Medians were determined for control samples at differentgestation ages. Since all medians were 0 (<0.5 ng/ml), no relationshipwas apparent between the level of free β-subunit and gestational age.ROC analysis was used to assess the effectiveness of measurementsindependent of arbitrary cut-off levels. The area under the ROC curvewas 0.68, indicating 68% discrimination between samples. Therelationship between free β-subunit levels in the washes and in theoriginal urine samples were determined. No correlation was observed(r²<0.5), indicating that they are independent variables. Resultsindicate a higher proportion of non-Con A binding molecules, oroligosaccharide variant molecules in Down syndrome pregnancies.

β-core fragment has degraded N-linked oligosaccharides containing 2.0N-acetylglucosamine and 0.5 fucose residues for 3.0 mannose residues(Blithe, D. L., et al., Endocrinology 125: 2267-2272 (1989) and Endo,T., et al., Endocrinology 130: 2052-2058 (1992)). Using a combination ofacetone and ethanol extraction, and gel filtration, blue-Sepharose™ (achromatography gel like Blue Dextran Sepharose® made by another company)and DEAE-Sepharose™ (a diethylaminoethl agarose chormatography gel)chromatography, β-core fragment was purified from 2 second trimesterDown syndrome urines and from 2 control preparations. N-terminal peptidesequence analysis (15 rounds) was performed. The control samplescontained 2.1 and 2.0 N-acetylglucosamine, and 0.6 and 0.5 fucoseresidues for 3.0 mannose residues, consistent with compositionsindicated by Blithe, et al., and Endo, et al., cited above. The Downsyndrome samples had different compositions, 3.5 and 3.2N-acetylglucosamine, and 0.8 and 1.4 fucose residues for 3.0 mannoseresidues. This suggested more complex oligosaccharide structures onβ-core fragment from Down syndrome.

Example 3

This example illustrates an immunoassay for Down syndrome pregnancies.

hCG preparations from individual normal pregnancy (6 women),hydatidi-form mole (3 women), and choriocarcinoma (4 women) wereisolated and purified from the urine of pregnant women as previouslydescribed (Kardana, A., et al. Endocrinology 129: 1541-1550 (1991)).Briefly, hCG was extracted from the urines by acetone and then ethanolprecipitations, followed by size-exclusion chromatography with Sephacry™S-200 (a chromatography gel disigned for the separation of proteinaccording to molecular size), ion exchange chromatography usingDEAE-Sepharose™ and, again, by size-exclusion chromatography with highresolution Sephacryl™ S-200. During chromatography procedures, attentionwas given to recovering all hCG fractions.

The peptide sequence and N- and O-linked oligosaccharide structures ofthe various hCG forms were determined as described previously (Elliott,M., et al., Endocrine J., vol. 7, 1997; see also Cole, L. A., andBirken, S., Mol. Endocrinol. 2: 825-830 (1988)). Monoclonal antibodiesto non-nicked hCG, intact hCG, free β-subunit, β-core fragment, andchoriocarcinoma hCG with hyperglycosylated N- and O-linkedoligosaccharide (hCG batch C5) were prepared using the above hCGpreparations following standard procedures (Ausubel, F. M., et al.,Short Protocols in Molecular Biology, 2 nd ed., 1992, unit 11). Briefly,mice were immunized with an hCG sample, given a second imununization inabout three weeks, and their spleens harvested after their bloodantibody levels showed an adequate response to the sample. The cellswere fused with myeloma cells, and hybridoma cells lines were obtained.Use of these monoclonals for the analysis of pregnancy urine wasreported at the Third World Conference on Early Pregnancy, Oct. 3-61996, in abstract number 66.

The monoclonals were employed to screen urine samples analyzed using theCon A lectin procedure of Example 2. One monoclonal raised againsthyperglycosylated hCG molecules (hCG batch C5) was denominated B152. Itrecognized <1% of normal hCG but recognized the same 60% of the samplesidentified as positive in the lectin assay.

The B152 monoclonal was then used to screen 139 urine samples obtainedfrom 130 pregnant women carrying normal fetuses and 9 women carryingDown's syndrome fetuses. Hyperglycosylated gonadotropin levels weredetermined in a two-step sandwich immunoassay using B152 as captureantibody, and peroxidaselabelled monoclonal antibody to hCGβ, batch 4001(Genzyme, San Carlos, Calif.) as tracer using the immunoassay methodspreviously described (Elliott, et al., cited above, see also Cole, L.,et al., J. Clin. Endocrinol. Metab. 76: 704-710 (1993)). Purehyperglycosylated gonadotropin (batch C7 described in the Cole, et al.,1988 paper cited above) was used to standardize the assay. Regular hCG(all forms of hCG dimer) levels were determined in a two-step sandwichassay using NIH CR127 hCG as a standard. Regular hCG andhyperglycosylated gonadotropin levels values are normalized tocreatinine concentration as described in Example 1.

Data from samples collected between 11 to 19 weeks of pregnancy are setout in the graph in FIG. 4, wherein  represents normal sample values,and ◯ represents the Down syndrome values. Results were analyzed usingstandard statistical methods. The gestational age specific medians forthe 130 control samples all best fit a log Gaussian distribution,between the fifth and ninety-fifth centiles, for both unaffectedpregnancy and Down syndrome data (expressed as multiples of theunaffected sample median, MoM). To assess screening performance, mediansand log standard deviations (estimated by the 10th to 90th centiledifference of the log MoM values, divided by 2.56) were determined, andthe observed detection rates recorded (i.e., proportion of Down syndromecases with levels exceeding 95th centile of unaffected pregnancies). Theformula for the median line was y=390(0.826^(x)), where y is the medianand x, the gestational age. The log median of the control samples was−0.007, and the log standard deviation was 0.45. All nine Down syndromecases exceeded the 70th centile of unaffected pregnancies. The medianhyperglycosylated gonadotropin level in Down syndrome was 4.4 multiplesof the unaffected median (log median=0.065). Six of the 9 Down syndromesamples (67%), including 2 of 3 first trimester and 4 of 6 secondtrimester cases, exceeded the 95th centile, and 3 of 9 samples (33%)exceeded the 100th centile of unaffected pregnancies.

Regular hCG levels were determined. Hyperglycosylated gonadotropinaccounted for 3.7% of the regular hCG molecules in normal pregnancy(mean 29 ng/mg and 781 ng/mg creatinine, respectively), and 11% of theregular hCG molecules in Down syndrome pregnancy samples (mean 193 ng/mgand 1690 ng/mg creatinine, respectively).

The results indicate that monoclonal B152 is probably recognizing hCGcarbohydrate variants, and that it can therefore be employed in animmunoassay for hyperglycosylated hCG in a method of the invention.

The above description is for the purpose of teaching the person ofordinary skill in the art how to practice the present invention, and itis not intended to detail all those obvious modifications and variationsof it which will become apparent to the skilled worker upon reading thedescription. It is intended, however, that all such obviousmodifications and variations be included within the scope of the presentinvention, which is defined by the following claims. The claims areintended to cover the claimed components and steps in any sequence whichis effective to meet the objectives there intended, unless the contextspecifically indicates the contrary.

The papers and patent cited above are expressly incorporated herein intheir entireties by reference.

What is claimed is:
 1. A method for screening for the presence orabsence of Down's syndrome in the fetus of a pregnant woman whichcomprises obtaining a biological test sample from the pregnant woman,contacting the sample with a reagent that detects hyperglycosylatedgonadotropin, removing excess reagent, and determining the presence ofDown's syndrome by observation of hyperglycosylated gonadotropin in thesample not observed in corresponding samples obtained from womencarrying normal fetuses.
 2. A method according to claim 1 wherein thetest sample is selected from the group consisting of urine, plasma andserum.
 3. A method according to claim 1 wherein the sample is obtainedduring the first or second trimester of pregnancy.
 4. A method accordingto claim 1 wherein observation of hyperglycosylated gonadotropin isdetermined using an antibody to at least one species of a chorionicgonadotropin population having aberrant carbohydrate moieties observedin Down's syndrome.
 5. A method according to claim 4 wherein a lectinspecific to the carbohydrate portion of hyperglycosylated gonadotropinis employed to separate species of chorionic gonadotropin havingaberrant carbohydrate moieties prior to immunoassay.
 6. A methodaccording to claim 1 wherein observation of hyperglycosylatedgonadotropin is determined using a lectin specific to the carbohydrateportion of Down's syndrome chorionic gonadotropin.
 7. A method accordingto claim 1 wherein observation of hyperglycosylated gonadotropincomprises an assay for monosaccharides in the sample.
 8. A methodaccording to claim 6, wherein the lectin comprises concanavalin A.
 9. Amethod according to claim 1, which comprises a comparison of thephysical properties of the hypenglycosyated gonadotropin population inthe test sample with a corresponding sample containing a normalchorionic gonadotropin population.
 10. A method according to claim 9wherein the comparison is made using a method selected from the groupconsisting of chromatography, electrophoresis, isoelectric focussing,and combinations of these techniques.
 11. A method for screening for thepresence or absence of Down's syndrome in the fetus of a pregnant womanwhich comprises: (a) obtaining a biological test sample from thepregnant woman, wherein the sample is selected from the group consistingof saliva, urine, plasma, and serum; (b) assaying for hyperglycosylatedgonadotropin in the test sample using an assay selected from the groupconsisting of carbohydrate analysis, immunoassay using specificantibodies to hyperglycosylated gonadotropin molecules, and acombination of carbohydrate analysis and immunoassay, and (c)determining the presence of Down's syndrome by observation ofhyperglycosylated gonadotropin in the sample not observed incorresponding samples obtained from women carrying normal fetuses.
 12. Amethod according to claim 11 wherein said sample is urine or serum andthe hyperglycosylated gonadotropin comprises hyperglycosylated hCG andβ-core fragment.
 13. A method according to claim 11 wherein the sampleis obtained during the first trimester.
 14. A method according to claim11 wherein the sample is obtained during the second trimester.
 15. Amethod according to claim 11 which employs an antibody to at least onespecies of a population having aberrant carbohydrate moieties observedin Down's syndrome.
 16. A method according to claim 15 wherein saidassaying step comprises an ELISA.
 17. A method according to claim 11wherein the assay employs a lectin specific to a variant carbohydrateportion of Down's syndrome gonadotropin.
 18. A method according to claim11, wherein the carbohydrate analysis is selected from the groupconsisting of chromatography, electrophoresis, isoelectric focussing,and combinations thereof.
 19. A method to according to claim 2 whereinthe test sample is urine.
 20. A method according to claim 11 wherein thetest sample is urine.